1. Field of the Invention
This invention relates to an in vitro method for enhancing the potential of oocytes to be fertilized.
2. Description of Related and Background Art
Inhibin and activin are members of a family of growth and differentiation factors. The prototype of this family is transforming growth factor-beta (TGF-.beta.). Derynck et al., Nature, 316: 701-705 (1985); Ying et al., Biochem. Biophys. Res. Commun., 135: 950-956 (1986). Other members of the TGF-.beta. family include the Mullerian inhibitory substance, the fly decapentaplegic gene complex, and the product of Xenopus Vg-1 mRNA.
Inhibin is a glycoprotein produced by diverse tissues, including the gonads, pituitary, brain, bone marrow, placenta, and adrenal gland. It was initially identified by its ability to inhibit the secretion of follicle stimulating hormone (FSH) by the pituitary. De Jong and Sharpe, Nature, 263: 71-72 (1976); Schwartz and Channing, Proc. Natl. Acad. Sci. USA, 74: 5721-5724 (1977). Such preferential regulation of the gonadotropin secretion has generated a great deal of interest and prompted many laboratories in the past fifty years to attempt to isolate and characterize this substance from extracts of testis, spermatozoa, rete testis fluid, seminal plasma, and ovarian follicular fluid using various bioassays. Rivier et al., Biochem. Biophys. Res. Commun., 133:120 (1985); Ling et al., Proc. Natl. Acad. Sci. USA, 82: 7217 (1985); Fukuda et al., Mol. Cell Endocrinol., 44:55 (1985). The structure of inhibin, characterized from several species, consists of two disulfide-linked subunits: an .alpha. and either a .beta..sub.A or a .beta..sub.B chain.
After the identification of inhibin, activin was shown to exist in follicular fluid as a naturally occurring substance. Activin was found to be capable of stimulating FSH release by rat anterior pituitary cells. Vale et al., Nature, 321: 776-779 (1986); Ling et al., Nature, 321: 779-782 (1986); DePaolo et al., Pro. Soc. Exp. Biol. Med., 198: 500-512 (1991); Ying, Endocrine Rev., 9: 267-293 (1988). Recombinant activin was also found to stimulate pituitary LH and FSH in the adult male macaque. McLachlan et al., Endocrinol., 125: 2787-2789 (1989). Activin consists of a homodimer or heterodimer of inhibin .beta. subunits, which may be .beta..sub.A or .beta..sub.B subunits. Vale et al., Recent Prog. Horm. Res., 44: 1-34 (1988). There is 95-100% amino acid conservation of .beta. subunits among human, porcine, bovine, and rat activins including the prepro region. The .beta..sub.A and .beta..sub.B subunits within a given species are about 64-70% homologous.
The inhibin heterodimers .alpha..beta..sub.A and .alpha..beta..sub.B ("Inhibin A" and "Inhibin B, " respectively) and the activin .beta..sub.A and .beta..sub.B homodimers ("Activin A" and "Activin B," respectively) have been identified in and purified from follicular fluid, and all these molecules have been cloned and their genes expressed. Mason et al., Biochem. Biophys. Res. Commun., 135:957 (1986); U.S. Pat. No. 4,798,885 issued Jan. 17, 1989; Mason et al., Molecular Endocrinol., 3: 1352-1358 (1989); Schwall et al., Mol. Endocrinol., 2: 1237-1242 (1988); Nakamura et al., J. Biol. Chem., 267: 16385-16389 (1992). The complete sequence of the .beta..sub.B subunit is published in Serono Symposium Publications, entitled "Inhibin- Non-Steroidal Regulation of Follicle Stimulating Hormone Secretion," eds. Burger et al., abstract by Mason et al., vol. 42, pp. 77-88 (Raven Press, 1987), entitled "Human Inhibin and Activin: Structure and Recombinant Expression in Mammalian Cells." The recombinant activin molecule has been shown to increase serum levels of FSH in rats when delivered by subcutaneous injection. Schwall et al., Endocrinol., 125: 1420-1423 (1989); Rivier and Vale, Endocrinol., 129: 2463-2465 (1991).
Activin and inhibin regulate the growth and functions of a variety of cell types. They may be involved in diverse biological processes including erythropoiesis, bone formation, placental and gonadal steroidogenesis, neuronal survival, and embryologic mesodermal induction. In addition, activin has an effect on follicular granulosa cell differentiation (Sugino et al., Biochem. Biophys. Res. Commun., 153: 281-288 [1988]), spermatogonial proliferation (Mather et al., Endocrinol., 127: 3206-3214 [1990]), erythroid differentiation (EP Publ. No. 210,461 published Feb. 4, 1987 [where the protein is called BUF-3]; Eto et al., Biochem. Biophys. Res. Commun., 142: 1095-1103 [1987] and Murata et al., Proc. Natl. Acad. Sci. USA, 85: 2434-2438 [1988] [where the activin is called EDF]; Yu et al., Nature, 330: 765-767 [1987] [where the activin is called FRP]), stimulation of insulin secretion by pancreatic islets (Totsuka et al., Biochem. Biophys. Res. Commun., 156: 335-339 [1988]), enhancement of proliferation of fibroblast (Hedger et al., Mol. Cell. Endocrinol., 61: 133-138 [1989]), stimulation of glucose production by hepatocytes (Mine et al., Endocrinology, 125: 586-591 [1989]), induction of a dose-dependent increase in inositol phosphates in rat parenchymal liver cells, an effect also seen with EGF (Mine et al., Biochem. Biophys. Res. Comm., 186: 205-210 [1992]), modulation of somatotroph functions (Billestrup et al., Mol. Endocrinol., 4: 356-362 [1990]), modulation of nerve cell differentiation (Schubert et al., Nature, 344: 868-870 [1990]; Hashimoto et al., Biochem. Biophys. Res. Comm., 173:193-200 [1990]), and mesoderm induction. Smith et al., Nature, 345: 729-731 (1990); Mitrani et al., Cell, 63: 495-501 (1990).
The expression of inhibin subunits, each encoded by a separate gene, was demonstrated in several tissues in addition to ovary. Woodruff et al., Molec. Endocrinol., 1: 561-568 (1987). Inhibin .alpha., .beta..sub.A, and .beta..sub.B mRNAs were detected in testis, placental, pituitary, adrenal, bone marrow, and brain tissues. Meunier et al., Proc. Natl. Acad. Sci. USA, 85: 247-251 (1988). The expression of the inhibin subunit mRNAs varied by several-fold in a tissue-specific manner, suggesting different functions for these proteins depending on their pattern of association and their site of production. Activin mRNA (.beta..sub.A and .beta..sub.B subunits), bioactivity, and immunoactivity have been reported to be produced by testicular Leydig cells from immature rat and pig. Lee et al., Science, 243: 396-398 (1989); Lee et al., in Serono Symposium Publications, entitled "The Molecular and Cellular Endocrinology of the Testis" eds. Cooke and Sharpe, Vol. 50 (Raven Press: New York, 1988), p. 21-27.
A new class of gonadal protein factors, named follistatin or FSH-suppressing protein (FSP), was isolated from side fractions derived from purifying porcine and bovine ovarian inhibins and activins. Ying, Endoc. Rev., 9: 267-293 (1988); Ling et al., "Isolation and characterization of gonadal polypeptides that regulate the secretion of follicle stimulating hormone," in Hodgen et al., eds., Non-Steroidal Gonadl Factors: Physiological Roles and Possibilities in Contraceptive Development, Jones Institute Press, Virginia, (1988), pp. 30-46. Follistatin was initially characterized by its ability to suppress FSH secretion from the pituitary. Thus, one biologic effect of follistatin is apparently similar to that of inhibin, but structurally the two proteins are quite different. Ueno etal., Proc. Natl. Acad. Sci. USA, 84: 8282-8286 (1987); Robertson et al., Biochem. Biophys. Res. Commun., 149: 744-749 (1987).
Follistatin is a glycosylated single-chain protein that is found in forms having molecular weights ranging from 31 to 39 kDa. All of these forms have similar amino acid compositions and identical amino-terminal amino acid sequences. The molecular cloning of cDNA with the gens of follistatin revealed two forms, a smaller molecular weight form and a larger form, which are generated by alternative splicing. The smaller form represents a carboxy-terminal truncated form of the larger precursor. For a review on follistatin and activin, see DePaolo et al., Proc. Soc. Exp. Biol. and Med., 198: 500-512 (1991). Follistatin is now thought to be an inhibin/activin binding protein.
In the human, growing preovulatory follicles and the corpus luteum secrete inhibin into the circulation in response to FSH stimulation. Lee and Gibson, Aust. J. Biol. Sci., 38: 115-120 (1985); McLachlan et al., Fertil. Steril., 48: 1001 (1987). Thus, inhibin-related peptides play important roles in the modulation of gonadal functions via a pituitary feedback loop. The secretion of inhibin by the corpus luteum has been proposed to suppress the concentration of FSH in the luteal phase of the cycle and hence the inhibition of follicular development. Baird et al., Ann. N. Y. Acad. Sci., 541: 153-161 (1988). However, recent data suggest that the corpus luteum does not secrete inhibin. Bramley et al., J. Endocrinol., 134: 341-352 (1992).
In primary cultures of rat testis cells and ovarian the calinterstitial cells, inhibin is reported to enhance androgen biosynthesis stimulated by luteinizing hormone (LH) (Hillier et al., Mol. Cell. Endocrinol., 75: R1-R6 [1991]), whereas activin suppresses androgen production. Hillier et al., J. Clin. Endocrinol. Metabol., 72: 1206-1211 (1991); Hsueh et al., Proc. Natl. Acad. Sci. USA, 84: 5082-5086 (1987). Other workers have been unable to repeat these observations in the male. deKretser and Robertson, Biol. Reprod., 40: 33-47 (1989).
Inhibitory effects of TGF-.beta. on Leydig cell steroidogenesis have also been described. Lin et al., Biochem. Biophys. Res. Commun., 146: 387 (1987); Fauser and Hsueh, Life Sci., 43: 1363 (1988); Avallet et al., Biochem. Biophys. Res. Commun., 146: 575 (1987). In granulosa cells, activin has been reported to inhibit (and TGF-.beta. to enhance) progesterone production. Ignotz and Massague, J. Biol. Chem., 261: 4337 (1986). In primary cultures of granulosa cells, activin and inhibin as well as TGF-.beta. were found to affect hormone synthesis and secretion, each in a different fashion. Adashi and Resnick, Endocrinology, 119: 1879 (1986); Ying et al., Biochem, Biophys. Res. Commun., 136: 969 (1986); Hutchinson et al., Biochem. Biophys. Res. Commun., 146: 1405 (1987); Mondschein et al., Endocinology, 123: 1970 (1988); Feng et al., J. Biol. Chem., 261: 14167 (1986). These molecules have both positive and negative effects on FSH-dependent granulosa cell function. Carson et al., J. Reprod. Fert., 85: 735-746 (1989). Also suggested is that individual members of the TGF-.beta./inhibin gene family regulate ovarian function, not only by direct action on follicle cells, but also indirectly by influencing the production rate of other members of that family. Zhiwen et al., Molecular and Cellular Endocrinology, 58: 161-166 (1988).
Activin and inhibin were reported to modulate growth of two gonadal cell lines, suggesting that these proteins may regulate proliferation as well as functions of gonadal cells. Gonzalez-Manchon and Vale, Endocrinology, 125: 1666-1672 (1989).
A review article postulates that inhibin is at least one of the factors that determines the number of follicles destined to ovulate, and that interference with the action of inhibin might contribute to the regulation of fertility. De Jong, Physiol. Rev, 68: 555 (1988). Many investigators have speculated that due to its FSH-inhibiting effect at the level of the pituitary, inhibin may be useful in male and female contraception. Sheth and Moodbidri, Adv. Contracept. 2: 131-139 (1986); Findlay, Fertil. Steril., 46: 770 (1986). Another author doubts that inhibin can inhibit spermatogenesis (citing Bremner et al., J. Clin. Invest., 68: 1044 [1981]), and states that inhibin might also have some direct stimulatory effects on spermatogenesis. Baker et al., Clin. Reprod. and Fert., 2: 161-174 (1983). It has now been shown that inhibin has a paracrine effect in stimulating ovarian follicular maturation. WO 91/10445.
When sheep are immunized with inhibin or the inhibin .alpha. chain, their ovulation rate is increased, due to the immunoneutralization of endogenous inhibin. Cummins et al., J. Reprod. Fertil., 77: 365 (1986); Henderson et al., J. Endocrinol., 102: 305-309 (1984); Forage et al., J. Endocrinol., 114: R1 (1987); Al-Obaidi et al., J. Reprod. Fert., 81: 403-414 (1987). The same effect has been observed in rats. Rivier and Vale, Endocrinology, 125: 152 (1989). In addition, Rivier and Vale suggest that increased FSH alone is sufficient to stimulate additional follicular growth and development, and the main mechanism through which treatment with anti-inhibin serum increases follicular development is through elevated plasma FSH levels. Other investigators reported that the administration of inhibin to sheep induces either anovulation or an increase in ovulation rate according to the scheme of treatment. Franchimont et al., Rev. fr. Gynecol. Obstet., 83: 607 (1988).
Activin is disclosed as useful for treating male infertility (see U.S. Pat. No. 5,166,190) and for treating polycystic ovarian disease when administered directly to the ovary. U.S. Pat. No. 5,102,868.
The modulation of the inhibin subunit mRNAs during the rat estrous cycle has been intensely studied. Woodruff et al., Science, 239: 1296 (1988). Only recently has the integrative feedback relationship between ovarian inhibin and activin and pituitary FSH been partially elucidated. Hasegawa et al., in Inhibin: Non-Steroidal Regulation of FSH Secretion, ed. J. Burger et al., 42: 119-133 (New York: Raven Press, 1987); Woodruff et al., Science, 239: 1296-1299 (1988). Briefly, the ovary produces low levels of inhibin on the evening of proestrus. Woodruff et al., Endocrinol., 2193-2199 (1989). This allows FSH to remain elevated throughout the morning of estrus (secondary FSH surge). Rivier et al., Science, 234: 205-208 (1986). The secondary FSH surge recruits a new set of follicles into the ovulatory pool and is responsible for the initiation of inhibin subunit mRNA expression. D'Agostino et al., Endocrinol., 124: 310-317 (1989). As a consequence of inhibin production, pituitary FSH secretion is downregulated. Inhibin mRNA levels increase in maturing follicles as they progress through the cycle. Follicles that become atretic (non-ovulatory and highly steroidogenic) have little or no inhibin mRNA. Woodruff et al., in Growth Factors and the Ovary, ed. Hirshfield, pp. 291-295 (New York: Plenum Press, 1989). Inhibin subunit mRNA accumulation climaxes on the afternoon of proestrus in healthy follicles simultaneously with the primary LH and FSH surges. Woodruff et al., Science, supra.
For maturation within their normal follicular environment, oocytes need to acquire meiotic competence but remain in meiotic arrest of prophase until stimulated to resume meiosis by the preovulatory gonadotropin surge. The oocyte becomes developmentally competent within a few hours of ovulation, having progressed to metaphase II (MII) and undergoing changes in metabolism, synthesis of proteins, and redistribution of cytoplasmic organelles. Sathananthan et al., "Maturation of the human oocyte," in Ultastructure of the Ovary, Familiari et al. (eds.) (Morwell, MA: Kluwer Academic Publishers, 1991), Chapter 2, pp. 29-43.
Oocyte maturation is necessary for successful in vitro fertilization (IVF). The stages of preovulatory maturation range from the immature germinal vesicle (GV) stage to MII of meoisis. The maturation includes both nuclear and cytoplasmic maturation of the germ cell.
Since the first successful IVF procedure in 1978, attempts have been made to employ drugs or hormones to induce multiple follicular and oocyte development. Thus, IVF pregnancies could result in women following the use of clomiphene citrate and human chorionic gonadotrophin (hCG) to induce multiple follicular development in endocrine normal patients. Trounson et al., Science, 212: 681-684 (1981). It is well established that the appropriate application of mixed exogenous gonadotropins FSH and LH has proven efficacious for ovulation induction or for multiple egg retrieval during IVF therapy. However, ovarian and oocyte stimulation in vivo by administration of exogenous gonadotropins is difficult to manage and costly due to the massive amounts of FSH/LH required for stimulation, and concerns exist over the possibility of increased ovarian cancer in gonadotropin-treated patients. Whittemore et al., Am. J. Epidemiol., 136: 1184-1203 (1992). Another agent of some use is FSH alone or in combination with a gonadotropin releasing hormone antagonist, as described in U.S. Pat. No. 4,845,077.
The treatments with FSH or a mixture of FSH and LH resulted in increased inhibin serum levels during the follicular and early luteal phase of the cycle. McLachlan et al., Lancet, 1: 1233-1234 (1986); Tsonis et al., J. Clin. Endocrinol Metab., 66: 915 (1988); Buckler et al., J. Endocrin., 122: 279-285 (1989); Tsuchiya et al., Fert. Steril., 52: 88 (1989). However, another investigator has found that at the time of ovulation neither inhibin activity nor follicular levels of steroid or gonadotropins are adequate criteria for predicting the performance of oocytes in an IVF protocol. Lefevre et al., Fert. Steril., 46: 325 (1986).
Activin A is reported to accelerate germinal vesicle breakdown (GVBD) of immature rat oocytes in vitro. Itoh et al., Biochem. Biophys. Res. Commun., 166: 1479-1484 (1990). Inhibin, on the other hand, reportedly inhibits meiotic maturation of rat oocytes as shown by the suppression of GVBD. O et al., Mol. Cell. Endo., 62: 307-311 (1989).
Routinely in IVF, oocyte collection is timed by the administration of hCG, unless there is an endogenous LH surge. Typically, maturation is completed by culturing the oocytes with their cumulus cells for 4-8 hours. Sathananthan and Trounson, Gamete Res., 5: 191-198 (1982); Trounson et al., J. Reprod. Fert., 64: 285-294 (1982). Various culture media are currently being used for both maturation and fertilization of oocytes. Trounson, in Clinical in Vitro Fertilization, Wood and Trounson, eds., 2nd ed. (London: Springer-Verlag, 1989), pp. 32-50. About 40% of oocytes are already at MII at the time of collection. If these MII oocytes age in culture before insemination, the chances of normal fertilization and embryo development of the oocytes are lessened considerably. Postmaturity is also associated with embryo mortality and chromosomal abnormalities. Osborne and Moor, J, Reprod. Fert. (Suppl.) 36: 59-72 (1988). oocytes at metaphase I (MI), which have undergone GVBD, are more likely to mature in time for insemination, and oocytes retrieved at the GV stage may still be immature at insemination.
It has been found that GV oocytes will complete meiotic maturation in Ham's F10 medium supplemented with follicular fluid recovered with mature oocytes. Cha et al., Fertil. Steril., 55: 109-113 (1991). These oocytes were fertilized at high rates, cleaved normally, and were capable of development to term (3 of 5 embryos were transferred). While it is thus possible that GV oocytes could be successfully cultured and fertilized to generate pregnancies, and while immature oocytes may undergo spontaneous maturation when released from their ovarian follicles, it would be desirable to enhance the rate and extent of maturation of immature oocytes in vitro so that a higher yield of mature oocytes could be accumulated for ultimate fertilization, such as by IVF. It would also be desirable to improve the quality of the oocytes that are being fertilized to increase the rate of success in fertilization and development.
Moreover, while the ethical advantage of cryopreserving human oocytes rather than embryos is evident, mature oocytes at MII are sensitive to cryoprotectants, cooling, and freezing. Trounson, "Embryo cryopreservation," in Clinical In Vitro Fertilization, Wood and Trounson, eds., 2nd ed. (Berlin Springer-Verlag, 1989), pp. 127-142; Trounson and Sathananthan, Human oocyte and embryo freezing, in Developments in Ultrastructure of Reproduction, Motta, ed. (New York: Alan R. Liss, 1989), pp. 355-366; Sathananthan et al., Hum. Reprod., 3: 968-977 (1988). Even brief cooling to room temperature from 37.degree. C. irreversibly disrupted spindle microtubules. Pickering et al., Fertil. Steril., 54: 102-108 (1990). Also, it was reported that fertilization of oocytes that were frozen and thawed caused dislocation of some of the chromosomes (Sathananthan and Trounson, "Effect of culture and cryopreservation on human oocyte and embryo ultrastructure and function," in Ultrastructure of Human Gametogenesis and Early Embryogenesis, Van Blerkom and Motta, eds. (Boston: Kluwer, 1989), pp. 181-199; Sathananthan et al., Gamete Res., 16: 343-354 [1987]) and polyploidy. Al-Hasani et al., Hum. Reprod., 2: 695-700 (1987). GV murine oocytes have been frozen and then cultured in vitro to maturation (Van Blerkom, Hum. Repord., 4: 883-898 [1989]); however, there is a need for a more efficient method to achieve maturation after cryopreservation.
Accordingly, it is an object of the present invention to increase the rate of maturation of oocytes in vitro before fertilization and embryo transfer.
It is another object to increase the degree of maturation of immature oocytes prior to fertilization and to enhance the quality of oocytes destined for fertilization so as to increase the chances of successful fertilization and subsequent development of a viable embryo.
It is yet another object to utilize what would otherwise be discarded oocytes in the context of an IVF program.
It is still another object to minimize the need for using expensive and time-consuming treatment with gonadotropins and minimize the time necessary for maturation in vitro.
It is a further object to achieve successful fertilization of cryopreserved oocytes, thereby circumventing ethical problems associated with the banking of fertilized human embryos.
It is a still further object to contribute significantly to the fertile oocyte pool required for the propagation of endangered non-human species such as primates or for development of disease models for medical research.
These and other objects will be apparent to one of ordinary skill in the art.